Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way

Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices

Flagellin pre-stimulation modulates the immune response to pathogens in pig airway epithelial cells

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Cytokine expression at interface: a longitudinal micro array

absen

Milk maturation temperature and time are key technological parameters to limit staphylococcal enterotoxin production during uncooked semi-hard cheese manufacture

International audienceThe influence of five technological parameters selected amongst uncooked semi-hard cheese-making practices, i. e. milk maturation temperature and time, stirring during maturation, curd stirring time and pressing time was examined on Staphylococcus aureus growth, enterotoxin gene expression and enterotoxin (SE) production during cheese manufacture. A fractional factorial experimental design was applied to perform 32 cheese batches independently inoculated at 103 cfu/ml of milk using four strains producing SEA, SEB, SEC or SED.The S. aureus population was found to exceed 105 cfu/g of cheese four hours after molding. SED was the only enterotoxin detected. It was produced in very low quantities that varied with the parameters studied. Early sed gene expression during cheese processing was correlated with SED detection in curd and cheese. Milk maturation temperature and time emerge as key technological parameters that control SED production. A response surface methodology was then carried out to further characterize the relationships between both factors and SE production in cheese and whey. Two SED-producing strains were used to perform two sets of ten cheese batches based on a central composite design of experiments at five levels. Predictive mathematical models were established.Increasing the temperature at the beginning of the cheese-making process was shown to increase SED production. Furthermore, we determined that the proportion of SED drained after molding from the curd in the whey depended only on the technological parameters. The two SED-producing strains showed similar trends of behavior but specific level of gene expression and enterotoxin production in response to the same set of milk maturation parameters

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae

International audienceHeme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection

TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium

International audienceExcessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs

Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese▿

Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production

Comparison of pathogenicity and intestinal immunity outcomes in infected pigs with French InDel and US non-InDel strains of porcine epidemic diarrhea virus

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Novel approaches boosting innate immunity against <em>Pseudomonas aeruginosa</em>

National audiencePseudomonas aeruginosa, an opportunistic gram-negative bacterium that rarely infects human lungs unless the host immune system has been impaired, is one of the main pathogens found in cystic fibrosis (CF) patients. P. aeruginosa infections in CF patients are difficult to treat, becoming chronic and contributing to exacerbated lung inflammation and respiratory failure. Modulation of innate immunity has been proposed as an alternative to improve defence against infections. This approach is particularly attractive in CF since exacerbated immune response is central to the pathogenesis of CF lung disease. Innate immunity in epithelial and immune cells can be stimulated through activation of Toll-like receptors (TLRs), the main family of pattern recognition receptors. Stimulation of TLR5 through flagellin-based interventions have demonstrated protective activity against several gram-negative bacteria (Salmonella sp., Burkholderia cepacia, Yersinia pseudotuberculosis), restoring immune-competence and promoting tissue repair processes. Here, we aimed to determine the effect of flagellin stimulation on the innate immune response against P. aeruginosa using an experimental pig model of lung infection. The pig model presents several advantages since swine and human lungs are similar in terms of anatomical, histological, biochemical, and physiological features. This is especially true in CF, where pigs lacking CFTR present a similar phenotype to what is typically observed in human patients. P. aeruginosa-infected pigs showed an acute neutrophilic response with an exacerbated release of neutrophil serine proteases that peaked 3-6 h post-infection (p.i.) leading to lung destruction and tissue hepatisation 24h p.i. When animals were pre-treated with flagellin 24h before the experimental infections, we observed a significant decrease in the expression of pro-inflammatory markers. In addition, a better lung status was observed on infected animals that had been pre-treated with flagellin compared to infected controls. The effect of flagellin pre-treatment on immune response to P. aeruginosa infection was confirmed using ex-vivo and in vitro models of lung epithelium from CFTR-/- pigs. In conclusion, our data point to a modulatory role of flagellin pre-treatment on the immune response to P. aeruginosa. This approach may have a therapeutic potential to improve inflammatory manifestations of CF